JPWO2015129764A1 - Method for producing cell population with high purity of target cell - Google Patents

Abstract

The present invention includes (1) a step of changing a trait of a contaminating cell in a cell population including the target cell and the contaminating cell, and (2) removing the contaminating cell based on the changed trait and / or the purpose. A method of producing a cell population having a high purity of a target cell, the step of collecting cells, a cell population having a high purity of the target cell produced by the method, and a step of culturing the cell population in a sheet form , A method for producing a sheet cell culture, a sheet cell culture containing the cell population, a pharmaceutical composition comprising an active ingredient selected from the group consisting of the cell population and the sheet cell culture, and the cell population And a method of treating a disease in the subject, comprising the step of administering an effective amount of a sheet-shaped cell culture or pharmaceutical composition to the subject in need thereof.

Description

  The present invention relates to a method for producing a cell population with high purity of target cells, a cell population obtained by the method, a pharmaceutical composition containing the cell population, and the like.

  In recent years, attempts have been made to transplant various cells in order to repair damaged tissues and the like. For example, fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, etc. have been tried to repair myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction. (Non-Patent Document 1).

As part of such attempts, cell structures formed using scaffolds and sheet-shaped cell cultures in which cells are formed into sheets have been developed (Patent Document 1).
For the application of sheet cell culture to the treatment, use of cultured epidermis sheet for skin damage caused by burns, use of corneal epithelial sheet cell culture for corneal injury, oral mucosa sheet for endoscopic resection of esophageal cancer Studies such as the use of cell cultures are ongoing.

  Cells used for cell transplantation are usually isolated from the tissue to be transplanted in order to avoid adverse events such as rejection, but the tissue often contains cells other than the target cell. When cells obtained from the tissue are cultured without purifying the target cells, cells other than the target cells proliferate, and the ratio of the target cells to the cells used for transplantation is low. However, even if cells other than the target cells are transplanted, the therapeutic effect is not as good as when the target cells are transplanted. Therefore, if the purity of the target cells is low, there is a possibility that sufficient therapeutic effects cannot be obtained even if cells obtained from tissues are transplanted.

  For example, when separating skeletal myoblasts from skeletal muscle tissue, the skeletal muscle tissue is immersed in a proteolytic enzyme solution for a predetermined time as a measure for increasing the proportion of target cells contained in the cell population separated from the tissue. After the enzyme treatment solution obtained by performing the enzyme treatment is discarded, a method of recovering cells contained in the enzyme treatment solution obtained by performing the enzyme treatment by dipping again in the proteolytic enzyme solution for a predetermined time, etc. Known (Patent Document 2).

Special table 2007-528755 gazette JP 2007-89442 A

Haraguchi et al., Stem Cells Transl Med. 2012 Feb; 1 (2): 136-41

  An object of the present invention is obtained by a method for producing a cell population having a high purity of a target cell from a cell population containing the target cell and a contaminated cell, in particular, a method for easily and efficiently producing such a cell population. The object is to provide a cell population with high purity of the target cell, a pharmaceutical composition containing the cell population, and the like.

  While the present inventor is diligently researching to solve the above problems, in the cell population including the target cell and the contaminating cell, the trait of the contaminating cell is changed, and the contaminating cell is removed based on the changed trait, In addition, the inventors have found that by collecting target cells, a cell population having high target cell purity can be produced, and the present invention has been completed.

That is, the present invention relates to the following.
<1> (1) a step of changing the character of the contaminating cell in a cell population including the target cell and the contaminating cell; and (2) removing the contaminating cell based on the changed character and / or the target cell. A method for producing a cell population having a high purity of a target cell, comprising a step of collecting.
<2> The method according to <1> above, wherein the contaminating cells are fibroblasts.
<3> The method according to <1> or <2> above, wherein the character is selected from the group consisting of cell shape, size, specific gravity, surface charge, adhesion ability, and marker.
<4> The method according to any one of <1> to <3>, wherein the target cell is a skeletal myoblast or a mesenchymal stem cell.

<5> A cell population produced by the method according to any one of <1> to <4> above, wherein the target cells have high purity.
<6> A method for producing a sheet-shaped cell culture, comprising the step of culturing the cell population according to <5> above in a sheet shape.
<7> A sheet-shaped cell culture comprising the cell population according to <5> above.
<8> A pharmaceutical composition comprising an active ingredient selected from the group consisting of the cell population according to <5> and the sheet-shaped cell culture according to <7>.
<9> The pharmaceutical composition according to <8> above, for treating a disease associated with a tissue abnormality.
<10> Subject requiring an effective amount of the cell population according to <5> above, the sheet-shaped cell culture according to <7> above or the pharmaceutical composition according to <8> or <9> above A method of treating a disease in said subject comprising administering to the subject.

  The present invention makes it possible to increase the purity of target cells contained in a cell population containing a plurality of types of cells collected from a living body, increase the therapeutic effect by cell transplantation, etc., and produce a cell composition used for such treatment. Increase efficiency. In addition, by selecting cell size, specific gravity, surface charge, etc. as the character to be changed, it becomes possible to use a simple method such as filtration and centrifugation that can process a large amount of cells at once. It can be purified efficiently.

FIG. 1 is a diagram showing the basic concept of the present invention.

  Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are hereby incorporated by reference in their entirety.

  One aspect of the present invention is (1) a step of changing a character of a contaminating cell in a cell population containing a target cell and a contaminating cell, and (2) removing the contaminating cell based on the changed character, and / or And a method for producing a cell population having a high purity of the target cell (hereinafter, sometimes abbreviated as “cell population production method”), including a step of collecting the target cell.

  In the present invention, target cells and contaminating cells can be arbitrarily set according to the use of the obtained cell population. Since the cell population containing the target cell and the contaminating cell is typically prepared from a tissue collected from a living body, the target cell and the contaminating cell are typically selected from cells existing in the same tissue. For example, since a cell population prepared from skeletal muscle tissue is mainly composed of skeletal myoblasts and fibroblasts, one of these can be a target cell and the other can be a mixed cell. Accordingly, in one embodiment of the present invention, the target cell is a skeletal myoblast and the contaminating cell is a fibroblast, and in another embodiment of the present invention, the target cell is a fibroblast and the contaminating cell is a skeletal myoblast. . In addition, since the cell population prepared from bone marrow tissue is mainly composed of mesenchymal stem cells, hematopoietic stem cells, blood progenitor cells, fibroblasts, etc., any one or more of these cells are targeted cells, others Can be used as a mixed cell. Therefore, in one embodiment of the present invention, without limitation, for example, even if the target cell is a mesenchymal stem cell, and the contaminating cells are hematopoietic stem cells, blood precursor cells, and fibroblasts, the target cell is mesenchymal. Stem cells, hematopoietic stem cells, and contaminating cells may be blood precursor cells and fibroblasts.

  Target cells and contaminating cells can also be selected from a part of cell types existing in living tissue. That is, it is not necessary to distribute all the cell types present in the living tissue into target cells and contaminating cells in the present invention, and cell types that can be easily separated from the target cells without changing the character are pre-treated by pretreatment. The target cells and contaminating cells can be selected from the cell types remaining after the pretreatment. Examples of the pretreatment include, but are not limited to, filtration (for example, filtration through a filter, a microchannel, etc.), centrifugation (for example, density gradient centrifugation, isodensity centrifugation, countercurrent centrifugation (Countercurrent centrifugal elutriation): CCE), etc.), and separation of adherent cells and suspension cells by adhesion culture. More specifically, for example, when producing a cell population having high purity of mesenchymal stem cells, which are target cells, from bone marrow tissue, first, floating cells contained in bone marrow tissue (for example, hematopoietic stem cells, blood progenitor cells, blood cells) Cells, etc.) are removed by pretreatment such as adhesion culture, and fibroblasts that are mainly contained in the remaining adherent cell population and are difficult to separate from mesenchymal stem cells are contaminated cells (ie, traits) in the present invention. Cell can be selected. Therefore, the cell population production method of the present invention may further include a step of removing cells other than the target cell and the contaminating cell from the tissue collected from the living body before the step of changing the character of the contaminating cell.

When the cell population is used for medical purposes (including but not limited to, for example, treatment of diseases, transplantation medicine, regenerative medicine, etc.), the target cells are not limited, and are used for treatment of blood diseases, for example. Hematopoietic stem cells, immune cells such as lymphocytes used for immunotherapy, dendritic cells, cells capable of forming a sheet-like cell culture, such as myoblasts (for example, skeletal myoblasts), mesenchymal stem cells ( Bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue, endometrium, placenta, umbilical cord blood, etc.), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, synoviocytes, cartilage Cells, epithelial cells (eg, oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells, etc.), hepatocytes (eg, liver parenchymal cells, etc.), pancreatic cells (eg, , Islet cells, etc.), kidney Cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, may be selected from skin cells or the like. In this case, the contaminating cells are selected from cells other than the target cells present in the tissue in which the target cells are present, in particular, cells that are difficult to separate from the target cells and have a high proliferation ability and / or a high ratio in the tissues be able to.
In one embodiment of the present invention, the contaminating cells are fibroblasts. In one embodiment of the present invention, the target cell is a myoblast, preferably a skeletal myoblast. In a preferred embodiment of the present invention, the target cell is a skeletal myoblast and the contaminating cell is a fibroblast. In another embodiment of the present invention, the target cell is a mesenchymal stem cell. In a preferred embodiment of the present invention, the target cell is a mesenchymal stem cell, and the contaminating cell is a fibroblast.

  As the trait of the contaminated cell to be changed, one or more known arbitrary traits can be selected, but the trait after the change is preferably useful for separation from the target cell. Examples of such traits include, but are not limited to, cell size, specific gravity, surface charge, presence / absence of marker expression, adhesion ability, proliferation ability, chemotaxis and the like. The alteration of the character can be performed by any known technique or a combination thereof. For example, it is known that one cell type can be converted to another cell type by introducing a gene such as a transcription factor or administering a drug (for example, Nizzardo et al., Cell Transplant. 2013; 22 (6): 921 -44, Zhu et al., Cell Reprogram. 2012 Apr; 14 (2): 99-105). Therefore, the step of changing the trait of the contaminating cell in the present invention includes using this technique to convert the contaminating cell into a cell type that can be easily separated from the target cell.

  For example, a technique for converting fibroblasts into adipocytes, megakaryocytes, platelets, hepatocyte-like cells, macrophage-like cells, nerve cells, cardiomyocytes, etc. is known (for example, the above-mentioned Nizzardo et al., 2013 Zhu et al., 2012, Ono et al., Blood. 2012 Nov 1; 120 (18): 3812-21, etc.), fibroblasts as contaminating cells are transformed into desired target cells such as skeletal myoblasts or Transformed into any of these cells that have different traits from mesenchymal stem cells, and the contaminated cells after the transformation are separated and removed from skeletal myoblasts or mesenchymal stem cells based on the difference in traits, and Alternatively, skeletal myoblasts or mesenchymal stem cells can be separated and removed from the contaminating cells after conversion. For example, the cell diameter of skeletal myoblasts is about 11-18 μm, the cell diameter of megakaryocytes is about 35-160 μm, the cell diameter of platelets is about 2-4 μm, the cell diameter of hepatocytes is about 20-30 μm, Since the cell diameter of macrophages is about 25-50 μm, if fibroblasts are converted into these cells, they can be separated from skeletal myoblasts using the difference in cell size. In addition, since adipocytes have a specific gravity different from that of skeletal myoblasts or mesenchymal stem cells, if fibroblasts are converted to adipocytes, the difference in specific gravity of the cells is used to convert skeletal myoblasts or mesenchymal cells. Can be separated from stem cells.

  The conversion of fibroblasts into adipocytes is not limited and is, for example, PPARγ (Tontonoz et al., Cell. 1994 Dec 30; 79 (7): 1147-56), C / EBPα (Freytag et al., Genes Dev. 1994 Jul 15; 8 (14): 1654-63), introduction of factors such as ADD1 / SREBP-1 (Kim and Spiegelman, Genes Dev. 1996 May 1; 10 (9): 1096-107), It can be carried out by treatment with a combination of a TGFβ signaling inhibitor (eg, a TGF-βRI kinase inhibitor such as SB431542) and a ROCK signaling inhibitor (eg, thiazobibin) (Zhu et al., 2012). The conversion of fibroblasts into hepatocyte-like cells is not limited, for example, a combination of introduction of Gata4, Hnf1α and Foxa3 and inactivation of p19Arf (Huang et al., Nature. 2011 May 11; 475 ( 7356): 386-9), introduction of Hnf4α and Foxa1, Foxa2 or Foxa3 (Sekiya and Suzuki, Nature. 2011 Jun 29; 475 (7356): 390-3). The conversion of fibroblasts into macrophage-like cells is not limited, for example, PU. 1 and C / EBPα or C / EBPβ (Feng et al., Proc Natl Acad Sci USA 2008 Apr 22; 105 (16): 6057-62) and the like. Conversion of fibroblasts into megakaryocytes and platelets can be performed without limitation, for example, by introduction of p45NF-E2, Maf G and Maf K (Ono et al., 2012). The conversion of fibroblasts into neurons is not limited, for example, introduction of Asc11, Brn2 and Mytll, introduction of Asc11, Brn2, Mytll and NeuroD1, miR-9 / 9, miR-124, NeuroD2, Asc11 and Introduction of Mytll, introduction of Mash1, Nurr1 and Lmx1a, introduction of Asc11, Pitx3, Lmx1a, Nurr1, Foxa2 and EN1, Asc11, Brn2, Nyt11, Lhx3, Hb9, Is11, Ngn2 and NEUROD1 et al. 2013). Conversion of fibroblasts into cardiomyocytes can be performed by introduction of Gata4, Tbx5 and Mef2c, introduction of Oct4, Sox2, Klf4 and c-Myc (Nizzardo et al., 2013).

  Other techniques for changing the character of the contaminated cell include, but are not limited to, expression or suppression of a desired marker, conversion to iPS cells, and subsequent differentiation induction into desired cells. Examples of the desired marker include, but are not limited to, cell surface markers, fluorescent proteins such as GFP, and the like. Marker expression or expression suppression can be performed by any known technique. Examples of the marker expression method include introduction of a nucleic acid encoding the marker into a contaminated cell. The introduction of the nucleic acid is not limited, and for example, calcium phosphate method, lipofection method, ultrasonic introduction method, electroporation method, particle gun method, vector (for example, adenovirus vector, adeno-associated virus vector, retrovirus vector, etc. ) Or a microinjection method can be used. Examples of the marker expression suppression method include, but are not limited to, RNAi method and antisense method. RNAi methods may include introduction of RNAi molecules into contaminated cells, and antisense methods may include introduction of antisense nucleic acids into contaminated cells.

  Conversion to iPS cells can be performed by introducing genes such as OCT3 / 4, SOX2, KLF4, C-MYC, LIN28, NANOG, GLIS1, etc. (for example, OCT3 / 4, SOX2, KLF4 and C-MYC combinations, OCT3 / 4, SOX2, LIN28 and NANOG combinations, OCT3 / 4, SOX2, KLF4 and GLISI combinations, OCT3 / 4, SOX2 and KLF4 combinations, OCT3 / 4 and SOX2 combinations, etc.). Methods for conversion to iPS cells are well known in the art (eg, Miyazaki et al., Jpn J Clin Oncol. 2012 Sep; 42 (9): 773-9, Bayart and Cohen-Haguenauer, Curr Gene Ther. 2013 Apr; 13 (2): 73-92, etc.), any known conversion method or variations thereof can be used in the present invention.

  Methods for converting iPS cells to other cell types are also well known in the art, and any known conversion method or modification thereof can be used in the present invention for converting iPS cells to megakaryocytes or platelets. For example, a medium containing STEMspan-ACF, BMP4, VEGF and bFGF, then a medium containing STEM-diff APEL Medium, TPO, SCF, Flt-3 ligand, IL-3, IL-6 and heparin, and then STEMspan -A method of sequentially culturing cells in a medium containing ACF, TPO, SCF, IL-6, IL-9 and heparin (Feng et al., Stem Cell Reports. 2014 Nov 11; 3 (5): 817-31 As a method for converting iPS cells into hepatocyte-like cells, for example, the presence of factors such as activin A, Wnt3, FGF4, bFGF, BMP-2, KGF, HGF, and oncostatin M A method of sequentially culturing cells (Subba Rao et al., World J Gastroenterol. 2013 Jun 14; 19 (22): 3385-96) is known.

  As used herein, an RNAi molecule refers to any molecule that causes RNA interference, including but not limited to siRNA (small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA), ddRNA (DNA- directed RNA), piRNA (Piwi-interacting RNA), double-stranded RNA such as rasiRNA (repeat associated siRNA), and variants thereof. These RNAi molecules are commercially available or can be designed and produced based on known sequence information. Antisense nucleic acid as used herein includes RNA, DNA, PNA, or a composite thereof.

When a nucleic acid (for example, a nucleic acid encoding a marker, an RNAi molecule or a nucleic acid encoding the same) or a drug is applied to a contaminated cell, a delivery method specific to the contaminated cell can be used. Such delivery techniques include, for example, active targeting (Marcucci and Lefoulon, Drug Discov Today. 2004 Mar 1; 9 (5): 219-) using a targeting agent (such as a targeting ligand) specific to contaminating cells. 28, Torchilin, Eur J Pharm Sci. 2000 Oct; 11 Suppl 2: S81-91 etc.). Many targeting agents specific to specific cells are known. Active targeting allows nucleic acids and drugs to act only on contaminating cells without acting on the target cells. For example, the same action on both target cells and contaminating cells (for example, conversion to the same cell type) It is possible to use nucleic acids and drugs that cause The targeting agent specific to fibroblasts is not limited, but includes, for example, TE-7, which is a fibroblast-specific cell surface marker (for example, Rosendal et al., J Cell Sci. 1994 Jan; 107 (Pt 1): 29-37, Goodpaster et al., J Histochem Cytochem. 2008 Apr; 56 (4): 347-58 etc.), for example, antibody or antigen recognition against TE-7 Examples thereof include fragments of the antibody containing the site (for example, F (ab ′) ₂ , Fab ′, Fab, Fv, scFv fragments, etc.).

  When the nucleic acid is expressed in a contaminated cell and used, the nucleic acid can be placed under the control of a regulatory sequence specific to the contaminated cell for expression specific to the contaminated cell. By using this technique, the nucleic acid can be expressed only in the contaminating cell without being expressed in the target cell. For example, the same action (for example, conversion to the same cell type) on both the target cell and the contaminating cell. It is possible to use a nucleic acid that brings about Many regulatory sequences that are specific to a particular cell are known. Regulatory sequences that can be used for specific expression of nucleic acids in fibroblasts are not limited, and include, for example, the ST2 gene proximal promoter (Iwahana et al., Eur J Biochem. 1999 Sep; 264 (2) : 397-406), α11 integrin promoter (Lu et al., Matrix Biol. 2010 Apr; 29 (3): 166-76), FSP1 gene promoter (Okada et al., Am J Physiol. 1998 Aug; 275 (2 Pt 2): F306-14), proα2 (I) collagen enhancer (Bou-Gharios et al., J Cell Biol. 1996 Sep; 134 (5): 1333-44) and the like. By using such a regulatory sequence in combination with the active targeting, cell specificity and the like can be further improved.

  The trait of the contaminated cell to be changed may be one or a combination of two or more. For example, when converting contaminated cells to another cell type, multiple traits may be altered simultaneously. In addition, a plurality of markers can be expressed at the same time, or the expression of a plurality of markers can be suppressed.

  The removal of the contaminating cells or the collection of the target cells can be performed by any known technique depending on the target trait. For example, if the trait to be changed is cell size, remove contaminated cells or collect target cells using appropriate size filters (for example, nylon mesh filters), filtration through microchannels, centrifugation, etc. can do. When the character to be changed is cell specific gravity, it is possible to remove contaminated cells or collect target cells using centrifugation (eg, density gradient centrifugation, iso-density centrifugation, counter-flow centrifugation, etc.) it can. If the altered trait is a cell surface marker, remove contaminating cells or remove target cells using flow cytometry, affinity separation (eg, affinity column method, magnetic cell separation, immunopanning, etc.) Can be collected. When the character after the change is the expression of fluorescent protein, the contaminated cells can be removed or the target cells can be collected using a flow cytometry method or the like. When the character to be changed is a surface charge, contaminating cells can be removed or target cells can be collected using ion exchange chromatography, electrophoresis, dielectrophoresis, or the like. Among these, from the viewpoints of the ease of purification operation and the large amount of cells that can be purified at one time, it is preferable to change a trait selected from the group consisting of cell size, specific gravity, and surface charge.

  The above method may be applied only once or multiple times for the same sample. Moreover, when two or more techniques exist for the same character, only one of them may be applied, or two or more may be applied in combination. Furthermore, when there are a plurality of traits to be changed, even if removal of the contaminating cells or collection of the target cells is focused on any one of them, the removal of the contaminated cells or the purpose is focused on two or more. Cells may be collected.

  In the present invention, “the purity of the target cell is high” means higher than the purity of the target cell in a natural tissue. For example, when the purity in the natural tissue is 50%, the purity of the target cell after the method of the present invention is 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85 % Or more, 90% or more, 95% or more, 98% or more, or 99% or more, and most preferably 100%. The purity of the target cell can be determined using any known technique. As such a technique, for example, the target cell is labeled with an antibody specific to the same cell, and the number of positive cells bound by the antibody is divided by the total number of cells counted. The cells can be counted by microscopic observation of a specimen stained with a specific antibody, image analysis of a microscopic image, flow cytometry analysis of a cell population stained with a specific antibody, and the like. When the target cell is a skeletal myoblast, the marker specific to the cell is not limited, and for example, CD56, α7 integrin, myosin heavy chain IIa, myosin heavy chain IIb, myosin heavy chain IId (IIx) , MyoD, Myf5, myogenin, etc., when the target cell is a mesenchymal stem cell, the marker specific to the cell is not limited, and examples thereof include CD29, CD73, CD90, CD105, CD166, etc. .

  The cell population production method of the present invention may further include a step of culturing the collected target cells and a step of subcultured the cultured cells. Cell culture and passage can be performed using any known method. The cell population production method of the present invention may further include a step of introducing a gene into the collected cells. The gene to be introduced is not particularly limited as long as it is useful for the treatment of the disease to be treated, and may be a cytokine such as HGF, for example. The gene can be introduced by any known method such as calcium phosphate method, lipofection method, ultrasonic introduction method, electroporation method, particle gun method, adenovirus vector, retrovirus vector or other viral vector method, or microinjection method. Can be used.

  Another aspect of the present invention relates to a cell population produced by the cell population production method of the present invention and having high target cell purity (hereinafter sometimes abbreviated as “high purity cell population”). The high-purity cell population of the present invention is obtained by the cell population production method of the present invention, and has a high target cell purity. The degree of target cell purity is as described above. Since the high-purity cell population of the present invention contains target cells at a high ratio, it is useful as a source of the cells used for medical use and the like. The high purity cell population of the present invention is preferably sterile. Moreover, the high-purity cell population of the present invention may be attached to a culture vessel, suspended in a physiologically acceptable liquid, or may be cryopreserved.

Another aspect of the present invention is a method for producing a sheet-shaped cell culture comprising the step of culturing the high-purity cell population of the present invention in the form of a sheet (hereinafter sometimes abbreviated as “sheet-shaped cell culture production method”). Is).
In the present invention, the “sheet-shaped cell culture” refers to a sheet in which cells are connected to each other and is typically composed of one cell layer, but is composed of two or more cell layers. Including those that are made. The cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances. The intervening substance is not particularly limited as long as it is a substance that can connect cells at least physically (mechanically), and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, particularly derived from cells constituting the sheet-shaped cell culture. The cells are at least physically (mechanically) connected, but may be further functionally, for example, chemically or electrically connected.

  The sheet-like cell culture of the present invention preferably does not contain a scaffold (support) from the viewpoints of biocompatibility and high therapeutic effect. Scaffolds may be used in the art to attach cells on and / or within its surface and maintain the physical integrity of sheet-like cell cultures, for example, polyvinylidene difluoride ( PVDF) membranes and the like are known, but preferred sheet-like cell cultures of the present invention can maintain their physical integrity even without such a scaffold. In addition, the sheet-shaped cell culture of the present invention is preferably composed only of cells derived from the cells constituting the sheet-shaped cell culture, and does not contain other substances.

  The step of culturing the high-purity cell population of the present invention in a sheet form can be performed by any known technique. Such a method is not limited and is, for example, Patent Document 1, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011-150 172925 etc. are mentioned. The step of culturing the high-purity cell population in a sheet form may include a step of seeding the cell population on a culture substrate and a step of forming the seeded cell population into a sheet.

The culture substrate is not particularly limited as long as the cell population can form a sheet-shaped cell culture thereon, and includes, for example, containers of various materials, solid or semi-solid surfaces in containers, and the like. The container preferably has a structure / material that does not allow permeation of a liquid such as a culture solution. Examples of such materials include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Examples include acrylamide and metals (for example, iron, stainless steel, aluminum, copper, brass). The container preferably has at least one flat surface. Examples of such containers include, but are not limited to, cell culture dishes and cell culture bottles. Further, the container may have a solid or semi-solid surface therein. Examples of solid surfaces include plates and containers of various materials as described above, and examples of semi-solid surfaces include gels and soft polymer matrices. The culture substrate may be prepared using the above materials, or commercially available materials may be used. Preferable culture substrates include, but are not limited to, substrates having an adhesive surface suitable for the formation of sheet cell cultures. Specifically, a substrate having a hydrophilic surface, for example, a substrate coated with a hydrophilic compound such as polystyrene subjected to corona discharge treatment, collagen gel or hydrophilic polymer, and further, collagen, fibronectin, laminin , Substrates coated with an extracellular matrix such as vitronectin, proteoglycan and glycosaminoglycan, and cell adhesion factors such as cadherin family, selectin family and integrin family. Such base materials are commercially available (for example, Corning ^(R) TC-Treated Culture Dish, Corning, etc.).

The surface of the culture substrate may be coated with a material whose physical properties change in response to stimulation, for example, temperature or light. Examples of such materials include, but are not limited to, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (for example, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylate Amides), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethyl) Chloramide and the like), (meth) acrylamide derivatives having a cyclic group (for example, 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo -2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, 4- (1-oxo -2-methyl-2-propenyl) -morpholine), or a vinyl ether derivative (for example, methyl vinyl ether) homopolymer or copolymer, a temperature-responsive material, a light-absorbing polymer having an azobenzene group, triphenylmethane leucohydro Copolymer of vinyl derivative of oxide and acrylamide monomer, and spirobenzopyra It can be used to include N- and isopropyl acrylamide gels known, such as photoresponsive materials (e.g., JP-A-2-211865, see JP-2003-33177). By giving a predetermined stimulus to these materials, the physical properties such as hydrophilicity and hydrophobicity can be changed, and peeling of the sheet-like cell culture adhered on the material can be promoted. Culture dishes coated with a temperature-responsive materials are commercially available (e.g., UpCell of CellSeed Inc. ^(R)) can be used, these the production method of the present invention.

  The seeding of the cell population on the culture substrate can be performed by any known technique and condition. The seeding of the cell population on the culture substrate may be performed, for example, by injecting a cell suspension obtained by suspending the cell population in the culture solution into the culture substrate (culture vessel). For the injection of the cell suspension, an apparatus suitable for the operation of injecting the cell suspension, such as a dropper or a pipette, can be used.

  The step of forming the seeded cell population into a sheet can also be performed by any known technique and conditions. Non-limiting examples of such methods include, for example, Patent Document 1, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011-172925, and the like. It is described in. It is considered that the sheet formation of the cell population is achieved by the cells adhering to each other via an adhesion molecule or an intercellular adhesion mechanism such as an extracellular matrix. Therefore, the step of forming the seeded cells into a sheet can be achieved, for example, by culturing a cell population under conditions that form cell-cell adhesion. Such conditions may be any as long as cell-cell adhesion can be formed, but cell-cell adhesion can usually be formed under the same conditions as general cell culture conditions. A person skilled in the art can select optimal conditions according to the type of cell population to be seeded. In the present specification, the culture for forming a seeded cell population into a sheet may be referred to as “sheet culture”.

The cell culture medium used for the culture (sometimes simply referred to as “culture medium” or “medium”) is not particularly limited as long as it can maintain cell survival, but typically, amino acids, vitamins, electrolytes are used. Can be used. A culture solution based on a basal medium for cell culture may be used. Such a basal medium is not limited, for example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), F12 / DMEM, L15, SkBM, RITC80 -7 and the like are included. Many of these basal media are commercially available, and their compositions are also known.
The basal medium may be used in a standard composition (for example, as it is commercially available), or the composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes one in which one or more components are added, removed, increased or decreased.

The cell population can be cultured under conditions usually used in the art. For example, typical culture conditions include culture at 37 ° C. and 5% CO ₂ . Culturing can be performed in containers of any size and shape. The size and shape of the sheet-shaped cell culture can be adjusted by adjusting the size and shape of the cell adhesion surface of the culture vessel, or by placing a mold of the desired size and shape on the cell adhesion surface of the culture vessel, It can be arbitrarily adjusted by, for example, culturing cells therein.

  The sheet-shaped cell culture production method of the present invention may further include a step of collecting the sheet-shaped cell culture. The recovery of the sheet-shaped cell culture is not particularly limited as long as the sheet-shaped cell culture can be released (peeled) from the culture substrate serving as a scaffold while at least partially maintaining the sheet structure. Enzymatic treatment with an enzyme (for example, trypsin) and / or mechanical treatment such as pipetting can be performed. In addition, when a cell population is cultured on a culture substrate whose surface is coated with a material that changes its physical properties in response to stimulation, for example, temperature or light, a sheet-like cell culture is formed. Can also be liberated non-enzymatically.

A particularly preferred embodiment of the method for producing a sheet-shaped cell culture of the present invention includes the following steps:
(1) changing the character of the contaminating cell in a cell population containing the target cell and the contaminating cell collected from the subject;
(2) removing contaminating cells based on the altered trait and / or collecting target cells to obtain a cell population with high purity of the target cells;
(3) freezing the cell population;
(4) thawing the frozen cell population;
(5) culturing the cell population in a sheet form to form a sheet-like cell culture;
(6) A step of collecting the formed sheet-shaped cell culture.
Steps (1) to (2) and (5) to (6) have already been described above. In the above embodiment, the target cell is preferably a myoblast (particularly skeletal myoblast) or a mesenchymal stem cell, and the contaminating cell is preferably a fibroblast.

Step (3) can be performed by any known method. Examples of such a technique include, but are not limited to, subjecting the cell population in the container to a freezing means such as a freezer, a deep freezer, and a low-temperature medium (for example, liquid nitrogen). The temperature of the freezing means is not particularly limited as long as it is a temperature at which a part of the cell population in the container, preferably the whole can be frozen, but is typically 0 ° C. or lower, preferably −20 ° C. or lower, more preferably − 40 ° C. or lower, more preferably −80 ° C. or lower. The cooling rate in the freezing operation is not particularly limited as long as it does not significantly impair the viability and function of the cells after freezing and thawing. Typically, the cooling rate is from 1 to 5 until cooling begins at 4 ° C and reaches -80 ° C. The cooling rate is on the order of time, preferably 2 to 4 hours, especially about 3 hours. Specifically, for example, cooling can be performed at a rate of 0.46 ° C./min. Such a cooling rate can be achieved by providing a container containing a cell population directly or by storing it in a freezing container in a freezing means set to a desired temperature. The freezing treatment container may have a function of controlling the temperature lowering speed in the container to a predetermined speed. As such a freezing container, any known container such as BICELL ^(R) (Japan Freezer) can be used.

  The freezing operation may be performed while the cell population is immersed in a culture solution or physiological buffer solution. However, a cryoprotectant for protecting cells from freezing and thawing operations may be added to the culture solution, or the culture solution may be frozen. You may perform after performing the process of replacing with the cryopreservation liquid containing a protective agent. Therefore, the sheet-shaped culture production method of the present invention may further include a step of adding a cryoprotectant to the culture solution or a step of replacing the culture solution with a cryopreservation solution. When replacing the culture solution with a cryopreservation solution, if the solution in which cells are immersed during freezing contains an effective concentration of cryoprotectant, remove the culture solution before adding the cryopreservation solution. Alternatively, the cryopreservation solution may be added while leaving a part of the culture solution. Here, the “effective concentration” means that the cryoprotectant exhibits a cryoprotective effect without exhibiting toxicity, for example, the viability, vitality, and function of the cell after freeze-thawing compared to the case where the cryoprotectant is not used. This means a concentration that exhibits a decrease-suppressing effect. Such a concentration is known to those skilled in the art or can be appropriately determined by routine experimentation.

  The cryoprotectant is not particularly limited as long as it exhibits a cryoprotective action on cells, for example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, Polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, mannitol and the like. Cryoprotectants may be used alone or in combination of two or more.

  The concentration of the cryoprotectant added to the culture solution or the concentration of the cryoprotectant in the cryopreservation solution is not particularly limited as long as it is an effective concentration as defined above. It is 2 to 20% (v / v) with respect to the whole preservation solution. However, although outside this concentration range, alternative use concentrations known or experimentally determined for each cryoprotectant may be employed, and such concentrations are within the scope of the present invention.

  Step (4) can be performed by any known cell thawing technique, typically involving, for example, thawing a frozen cell population into a thawing means, eg, solid, liquid or gaseous, at a temperature above the freezing temperature. Although it is achieved by subjecting the frozen cell population to a medium (for example, water), a water bath, an incubator, an incubator, or the like, or immersing the frozen cell population in a medium (for example, a culture solution) at a temperature higher than the freezing temperature, It is not limited to this. The temperature of the thawing means or the immersion medium is not particularly limited as long as it is a temperature at which the cell population can be thawed within a desired time, but typically 4 to 50 ° C., preferably 30 to 40 ° C., more preferably 36 to 38 ° C. The thawing time is not particularly limited as long as it does not significantly impair the viability and function of the cells after thawing, but it is typically within 2 minutes, and in particular within 20 seconds can reduce the viability. It can be greatly suppressed. The thawing time can be adjusted, for example, by changing the temperature of the thawing means or the immersion medium, the volume or composition of the culture solution or cryopreservation solution at the time of freezing.

  The sheet-shaped cell culture production method may include a step of washing the cell population between step (4) and step (5). Washing of the cell population can be performed by any known technique, and typically includes, for example, a culture or physiological solution that contains or does not contain the cell population in liquid (eg, serum or serum components (such as serum albumin)). This is achieved by, but not limited to, suspending in a buffer or the like, centrifuging, discarding the supernatant, and collecting the precipitated cell population. In the step of washing the cell population, the suspension, centrifugation, and recovery cycle may be performed once or a plurality of times (for example, 2, 3, 4, 5, etc.). In one aspect of the invention, the step of washing the cell population is performed immediately after step (4) of thawing the frozen cell population.

  In one embodiment, all steps of the production method of the present invention (including the cell population production method and the sheet-like cell culture production method) are performed in vitro. In another embodiment, the production method of the present invention includes a step performed in vivo, including, but not limited to, collecting a cell population or a tissue serving as a source of the cell population from a subject. In one embodiment, the production method of the present invention is performed under aseptic conditions in all steps. In one embodiment, the production method of the present invention is performed so that the finally obtained cell population or sheet-shaped cell culture is substantially sterile. In one embodiment, the production method of the present invention is performed so that the finally obtained cell population or sheet-shaped cell culture is sterile.

Another aspect of the present invention relates to a sheet-shaped cell culture (hereinafter sometimes abbreviated as “sheet-shaped cell culture”) comprising the high-purity cell population of the present invention.
The sheet-like cell culture of the present invention has a high purity of target cells. The high purity is as described above for the high purity cell population of the present invention. The target cell is preferably a myoblast, particularly a skeletal myoblast, or a mesenchymal stem cell. The sheet-shaped cell culture of the present invention may be produced by the sheet-shaped cell culture production method of the present invention. The sheet-shaped cell culture of the present invention is preferably sterile. Since the sheet-shaped cell culture of the present invention contains target cells with high purity, the therapeutic effect and the like are higher than those of the sheet-shaped cell culture not including the high-purity cell population of the present invention.

  The high-purity cell population and sheet-shaped cell culture of the present invention are useful for the treatment of various diseases, particularly diseases associated with tissue abnormalities. Accordingly, in one aspect, the high purity cell population and sheet cell culture of the present invention are for use in the treatment of diseases associated with tissue abnormalities. Examples of the tissue to be treated include, but are not limited to, myocardium, cornea, retina, esophagus, skin, joint, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, middle ear, bone marrow, and the like. It is done. In addition, the disease to be treated is not limited, and for example, heart disease (eg, myocardial injury (myocardial infarction, cardiac injury), cardiomyopathy, etc.), corneal disease (eg, corneal epithelial stem cell exhaustion, cornea) Injury (heat / chemical corrosion), corneal ulcer, corneal opacity, corneal perforation, corneal scar, Stevens-Johnson syndrome, pemphigoid, etc., retinal diseases (eg retinitis pigmentosa, age-related macular degeneration) , Esophageal diseases (for example, prevention of esophageal inflammation / stenosis after esophageal surgery (esophageal cancer removal)), skin diseases (for example, skin damage (trauma, burn), etc.), joint diseases (for example, osteoarthritis, etc.) Cartilage disease (eg, cartilage damage), liver disease (eg, chronic liver disease), pancreatic disease (eg, diabetes), dental disease (eg, periodontal disease), kidney disease (eg, renal failure) Renal anemia, kidney Bone dystrophy), thyroid disease (eg, hypothyroidism), muscle disease (eg, muscle damage, myositis), middle ear disease (eg, otitis media), bone marrow disease (eg, leukemia, poor regeneration) Anemia, immunodeficiency diseases, etc.).

  The high-purity cell population and sheet-shaped cell culture of the present invention are useful for the above-mentioned diseases, for example, Patent Document 1, Non-Patent Document 1, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12 ): 3943-9, Ito et al., Tissue Eng. 2005 Mar-Apr; 11 (3-4): 489-96, Yaji et al., Biomaterials. 2009 Feb; 30 (5): 797-803, Yaguchi et al., Acta Otolaryngol. 2007 Oct; 127 (10): 1038-44, Watanabe et al., Transplantation. 2011 Apr 15; 91 (7): 700-6, Shimizu et al., Biomaterials. 2009 Oct; 30 (30): 5943-9, Ebihara et al., Biomaterials.2012 May; 33 (15): 3846-51, Takagi et al., World J Gastroenterol.2012 Oct 7; 18 (37): 5145-50 etc. Have been described.

  The high-purity cell population and sheet-shaped cell culture of the present invention can be applied to a tissue to be treated and used to repair and regenerate the tissue, but as a source of a physiologically active substance such as a hormone, It can also be transplanted to a site other than the tissue to be treated (eg, subcutaneous tissue) (eg, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Shimizu et al ., Biomaterials. 2009 Oct; 30 (30): 5943-9). It is also possible to fragment the sheet-like cell culture of the present invention into an injectable size and inject it into a site requiring treatment (Wang et al., Cardiovasc Res. 2008 Feb 1; 77 (3) : 515-24).

  The high-purity cell population and sheet-shaped cell culture of the present invention can be prepared by adding various additional components such as a pharmaceutically acceptable carrier, and the viability and engraftment of the high-purity cell population and / or the sheet-shaped cell culture. In addition, it may further contain a component that enhances the function and the like, another active component useful for the treatment of the target disease, and the like. Any known additional components can be used, and those skilled in the art are familiar with these additional components. In addition, the high-purity cell population and sheet-shaped cell culture of the present invention include components that enhance the viability, engraftment and / or function of the high-purity cell population and / or sheet-shaped cell culture, and treatment of target diseases. It can be used in combination with other active ingredients useful for.

Another aspect of the present invention relates to a pharmaceutical composition comprising an active ingredient selected from the group consisting of the high-purity cell population and sheet-shaped cell culture of the present invention.
In addition to the high-purity cell population and / or sheet-shaped cell culture of the present invention, the pharmaceutical composition of the present invention can be prepared by adding various additional components such as a pharmaceutically acceptable carrier, high-purity cell population and / or It may contain components that enhance the viability, engraftment and / or function of the sheet-shaped cell culture, other active ingredients useful for the treatment of the target disease, and the like. Any known additional components can be used, and those skilled in the art are familiar with these additional components. In addition, the pharmaceutical composition of the present invention is a component that enhances the viability, engraftment and / or function of a high-purity cell population and / or sheet-shaped cell culture, and other active ingredients useful for the treatment of the target disease. Etc. can be used together. In one aspect, the pharmaceutical composition of the present invention is for use in the treatment of diseases associated with tissue abnormalities. The tissues and diseases to be treated are as described above for the high-purity cell population and sheet-shaped cell culture of the present invention.

  Another aspect of the present invention is the high purity of the present invention comprising some or all of the elements used in the production of the high purity cell population, sheet cell culture or composition (eg, pharmaceutical composition) of the present invention. Kits (sets or packs) for producing cell populations, sheet-like cell cultures or compositions (eg, pharmaceutical compositions), etc., or for treating diseases (eg, diseases related to tissue abnormalities, etc.) (Hereinafter sometimes referred to as “production kit of the present invention”). In this specification, the terms “set” and “pack” are used interchangeably with “kit”, and the description relating to “kit” in this specification also applies to “set” and “pack”.

  The specific elements contained in the kit of the present invention will be apparent by referring to the above description relating to the production of the high-purity cell population, sheet-shaped cell culture or pharmaceutical composition of the present invention. Are not listed. In one embodiment, the kit of the present invention is not limited, for example, an agent for changing the character of a contaminated cell (for example, an agent for converting a cell type, an agent for expressing or suppressing a marker, agents for conversion to iPS cells, agents for inducing differentiation of iPS cells into other cell types, etc.), cell populations used to produce high purity cell populations (eg, cell populations isolated from living organisms), Cells used for culturing a sheet-shaped cell culture (for example, the high-purity cell population of the present invention), a culture solution, a culture dish, a washing solution, an agent used for purification of target cells and / or elimination of contaminating cells (for example, antibodies, Cleaning solutions, etc.), instruments (eg, pipettes, droppers, tweezers, beads, affinity columns, filters, etc.), high-purity cell populations, sheet cell cultures or compositions, etc. Instructions (e.g., instructions, medium recording information on the production method and use, for example, flexible disk, CD, DVD, Blu-ray disc, memory card, USB memory, etc.) may contain such.

  In a specific embodiment, the kit for producing a high-purity cell population of the present invention or a composition containing the same comprises an agent for altering the trait of contaminating cells, and purification of target cells and / or elimination of contaminating cells. Including the agent used for. In another specific embodiment, a kit for producing a high-purity cell population of the present invention or a composition comprising the same is provided, a cell population isolated from a living body, an agent for altering the trait of contaminating cells, and an object Contains agents used for cell purification and / or elimination of contaminating cells. In another specific embodiment, a kit for producing a sheet-shaped cell culture of the present invention or a composition containing the same includes the high-purity cell population of the present invention, a culture medium, and a culture dish. In another specific embodiment, the kit for producing the sheet-shaped cell culture of the present invention or the composition containing the same comprises a cell population isolated from a living body, an agent for changing the character of contaminating cells, a target cell And / or agents used for the purification and / or elimination of contaminating cells.

  Another aspect of the invention is a method of treating a disease in a subject comprising administering to a subject in need thereof an effective amount of a high purity cell population, sheet-like cell culture or pharmaceutical composition of the invention. The method includes (hereinafter may be abbreviated as “treatment method”). The tissues and diseases to be treated by the treatment method of the present invention are as described above for the high-purity cell population and sheet-shaped cell culture of the present invention. Further, in the treatment method of the present invention, a component that enhances the viability, engraftment and / or function of the high-purity cell population and / or cell culture, other active ingredients useful for the treatment of the target disease, etc. The high-purity cell population, sheet-shaped cell culture or pharmaceutical composition of the present invention can be used in combination.

  The treatment method of the present invention may further comprise the step of producing a high-purity cell population, sheet-shaped cell culture or pharmaceutical composition according to the production method of the present invention. The treatment method of the present invention provides a source of whole blood, cells or cells to produce a high purity cell population from a subject prior to the step of producing a high purity cell population, sheet-like cell culture or pharmaceutical composition. The method may further include collecting the tissue. In one embodiment, the subject from whom the whole blood and / or cells or tissue that is the source of cells is collected is the same individual as the subject receiving the high-purity cell population, sheet cell culture, or pharmaceutical composition. In another embodiment, the subject from whom the whole blood and / or cells or tissue that is the source of cells is collected is a separate species of the same type as the subject receiving the high-purity cell population, sheet-like cell culture, or pharmaceutical composition. is there. In another embodiment, the subject from whom whole blood and / or cells or tissue that is the source of cells is collected is an individual different from the subject receiving the high-purity cell population, sheet-like cell culture, or pharmaceutical composition. .

In the present invention, the subject may be healthy or afflicted with some disease, but when treatment of the disease is intended, the subject is typically afflicted with or affected by the disease. Means a subject at risk.
The term “treatment” is also intended to encompass all types of medically acceptable prophylactic and / or therapeutic interventions intended to cure, temporarily ameliorate or prevent disease. For example, the term “treatment” encompasses medically acceptable interventions for various purposes, including delaying or stopping the progression of the disease, regression or disappearance of the lesion, prevention of the onset of the disease or prevention of recurrence, etc. .

  In the present invention, the effective amount is, for example, an amount that can suppress the onset or recurrence of a disease, reduce symptoms, or delay or stop progression (for example, a high-purity cell population, a sheet-shaped cell culture, or a pharmaceutical composition). The number of cells contained in the product, the size and weight of the sheet-like cell culture, etc., and preferably the amount that prevents the onset and recurrence of the disease or cures the disease. In addition, an amount that does not cause adverse effects exceeding the benefits of administration is preferred. Such an amount can be appropriately determined by, for example, testing in laboratory animals such as mice, rats, dogs or pigs, and disease model animals, and such test methods are well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important index for determining the effective amount.

The high-purity cell population, sheet-shaped cell culture or pharmaceutical composition of the present invention is administered from various routes such as intravenous, intramuscular, subcutaneous, topical, intraarterial, intraportal, intraventricular, intraperitoneal, etc. Can do. In the case of a sheet-shaped cell culture or a pharmaceutical composition containing the same, the administration method typically includes direct application to tissues, but when a fragment of the sheet-shaped cell culture is used, an injection is used. It may be administered from various routes that can be administered by, for example, intravenous, intramuscular, subcutaneous, topical, intraarterial, intraportal, intraventricular, intraperitoneal, and the like.
The frequency of administration is typically once per treatment, but multiple administrations are possible if the desired effect is not obtained.

Examples Example 1: Purification of skeletal myoblasts by conversion of fibroblasts into adipocytes A cell suspension of a cell population isolated from skeletal muscle is 0.8 x 10 < ⁵ > / well. Inoculate to a cell culture plate and culture overnight in a CO ₂ incubator. 30 μL of serum-free medium was placed in a microtube, and 1.0 μg of a PPARγ-expressing adenovirus vector (AdRGD-PPARγ, Gastroenterology. 2003 May; 124 (5): 1315-24) was added thereto, followed by mixing by pipetting. Add 5 μL of HilyMax solution (manufactured by Dojindo Laboratories) and let stand at room temperature for 15 minutes to prepare AdRGD-PPARγ-HilyMax complex. AdRGD-PPARγ-HilyMax complex is added to the cultured cell population, and the plate is gently shaken. The cells are cultured for 24 hours in a CO ₂ incubator. The cell population was washed with PBS, centrifuged, seeded in an 80 cm ² flask at a density of ^{2 to} 3 × 10 ⁴ cells / cm ² , and 5 to 7 with 15 mL of medium (medium for adipocyte differentiation induction: Cosmo Bio). Incubate for days. After culturing, the culture surface is washed with PBS, and the cell population obtained by trypsin treatment is filtered with a mesh having a pore size of 70 μm (cell strainer: manufactured by Corning), and the cell population passing through the mesh is obtained as the target cell population. . The obtained cell population is labeled with an anti-CD56 antibody, and the proportion of CD56 positive cells (skeletal myoblast purity) is measured using a flow cytometer.

  Those skilled in the art will appreciate that many different modifications can be made without departing from the spirit of the invention. Accordingly, it is to be understood that the forms of the invention described herein are exemplary only and are not intended to limit the scope of the invention.

Claims (10)

(1) a step of changing the character of the contaminating cell in a cell population including the target cell and the contaminating cell; and (2) a step of removing the contaminating cell and / or collecting the target cell based on the changed character. A method for producing a cell population having a high purity of a target cell.   The method of claim 1, wherein the contaminating cells are fibroblasts.   The method according to claim 1 or 2, wherein the character is selected from the group consisting of cell shape, size, specific gravity, surface charge, adhesion ability, and marker.   The method according to any one of claims 1 to 3, wherein the target cell is a skeletal myoblast or a mesenchymal stem cell.   A cell population produced by the method according to claim 1, wherein the target cell has a high purity.   A method for producing a sheet-shaped cell culture, comprising the step of culturing the cell population according to claim 5 in a sheet form.   A sheet-shaped cell culture comprising the cell population according to claim 5.   A pharmaceutical composition comprising an active ingredient selected from the group consisting of the cell population according to claim 5 and the sheet-shaped cell culture according to claim 7.   The pharmaceutical composition according to claim 8 for treating a disease associated with a tissue abnormality.   Administering to the subject in need thereof an effective amount of the cell population of claim 5, the sheet cell culture of claim 7, or the pharmaceutical composition of claim 8 or 9, A method of treating a disease in a subject.